Project Abstract/Summary Potent inhibitors of the flavivirus membrane fusion mechanism pose an appealing option for treatment and prevention of infection. A deeper understanding of how the molecular rearrangements of the glycoprotein E mediates lead to membrane fusion and deposition into a cell of viral genetic material would greatly aid the design of therapeutics. Moreover, information about the binding modes of the current small molecule inhibitors that effectively prevent membrane fusion would facilitate design of improved and more-drug-like compounds. The aims of this proposal are: (i) to determine the structure of the full-length flavivirus E glycoprotein (including its transmembrane anchors) in its trimeric postfusion conformation and use this information to inform kinetic analyses of the membrane fusion process; (ii) to define the binding site of previously characterized small molecule inhibitors of flavivirus hemifusion in various conformational states along the fusion pathway. Flavivirus E glycoprotein facilitates membrane fusion between the cellular membrane of the endosome and viral membrane envelope. Upon reaching the low pH environment of an endosome, a series of conformational rearrangements occurs in which the E protein dimer present on the surface of a mature virus dissociates to a projecting monomer, allowing its ?fusion loop? to engage with the cellular membrane. The E protein monomers then trimerize and bring the two adjacent membranes together into one continuous membrane through two steps, a hemifusion intermediate step and a complete fusion (fusion-pore opening) step. The ?stem? of the E protein bridges the soluble ectodomain and the transmembrane anchor; its folding back drives membrane fusion. Previous attempts to structurally characterize the membrane interacting regions of the flavivirus E protein have failed because their hydrophobicity causes formation of soluble aggregates. Using cryoelection microscopy, I will determine the high-resolution structure of the full-length E protein in its trimeric, postfusion conformation. Information derived from the structure will then be used make mutants that lack membrane-fusion activity. Single particle membrane fusion assays between virus-like particles and supported lipid bilayers formed in microfluidic chambers will be carried out with these mutants to investigate the kinetics of the final membrane fusion step. Information provided by these mutational analyses will provide a foundation for the design of new classes of inhibitors. Efforts to create small molecule inhibitors of flavivirus membrane fusion have already been pursued by Harrison and collaborators; compounds exist that efficiently inhibit the hemifusion step. The site of binding of these compounds on the E glycoprotein is probably the so-called ?-octyl glucoside pocket, but definitive assignment and description of the binding pose are both still lacking. Both are needed for further medicinal chemistry. As these inhibitors bind to multiple states along the membrane fusion pathway, I will use cryoEM to determine the binding-site and pose of the inhibitor on several conformations of the E protein.